Bottom Line:
In contrast a majority of the oligomers at both early (1 hour) and late times (24 hours) traffic with a fluid-phase marker, dextran, to the same endocytotic compartments, the uptake of which is blocked by potent macropinocytotic inhibitors.This led to a significant increase in extra-cellular PM accumulation, in turn potentiating amylin toxicity in pancreatic cells.Our studies suggest that macropinocytosis is a major but not the only clearance mechanism for both amylin's molecular forms, thereby serving a cyto-protective role in these cells.

Affiliation: Department of Biological Sciences, The George Washington University, Washington, District of Columbia, United States of America.

ABSTRACTToxic human amylin oligomers and aggregates are implicated in the pathogenesis of type 2 diabetes mellitus (TTDM). Although recent studies have shown that pancreatic cells can recycle amylin monomers and toxic oligomers, the exact uptake mechanism and trafficking routes of these molecular forms and their significance for amylin toxicity are yet to be determined. Using pancreatic rat insulinoma (RIN-m5F) beta (β)-cells and human islets as model systems we show that monomers and oligomers cross the plasma membrane (PM) through both endocytotic and non-endocytotic (translocation) mechanisms, the predominance of which is dependent on amylin concentrations and incubation times. At low (≤ 100 nM) concentrations, internalization of amylin monomers in pancreatic cells is completely blocked by the selective amylin-receptor (AM-R) antagonist, AC-187, indicating an AM-R dependent mechanism. In contrast at cytotoxic (µM) concentrations monomers initially (1 hour) enter pancreatic cells by two distinct mechanisms: translocation and macropinocytosis. However, during the late stage (24 hours) monomers internalize by a clathrin-dependent but AM-R and macropinocytotic independent pathway. Like monomers a small fraction of the oligomers initially enter cells by a non-endocytotic mechanism. In contrast a majority of the oligomers at both early (1 hour) and late times (24 hours) traffic with a fluid-phase marker, dextran, to the same endocytotic compartments, the uptake of which is blocked by potent macropinocytotic inhibitors. This led to a significant increase in extra-cellular PM accumulation, in turn potentiating amylin toxicity in pancreatic cells. Our studies suggest that macropinocytosis is a major but not the only clearance mechanism for both amylin's molecular forms, thereby serving a cyto-protective role in these cells.

Mentions:
It is well documented that human amylin is toxic to pancreatic rat and human islet cells [4], [50], [60], [61]. However, the exact mechanism and endocytotic machinery regulating amylin turnover in pancreatic cells remain largely unknown. Using immunoconfocal microscopy, we investigated the roles of amylin receptor (AM-R) and endocytosis in the uptake and toxicity of human amylin in cultured pancreatic RIN-m5F and human islet cells. As human amylin is non-toxic at low (nM) concentrations and cytotoxic at higher (µM) concentrations [50], we examined the mechanism of amylin monomer and oligomer internalization at these two distinct concentrations, aiming to understand how cells deal with amylin overload. Hence, cells were incubated for 24 hours with low (100 nM) or high (10 µM) concentrations of freshly prepared human amylin and its intracellular/PM accumulation determined by quantitative immunoconfocal analysis (Figure 1, Figure S1) [4]. Prolonged incubation of cells with 100 nM human amylin allowed amylin accumulation both on the PM and in the perinuclear compartments (Figure 1A, top panel). Whole cell analysis (Figure 1A, top panel and graph) revealed that monomers were equally distributed between PM and intracellular compartments. Incubations of cells with 10 µM human amylin increased intracellular accumulations of monomeric amylin by ∼20% (Figure 1A, bottom panel and graph), indicating a saturable and possibly receptor dependent mechanism for amylin uptake. To confirm or refute a receptor dependent mechanism for amylin monomer uptake, cells were co-incubated with human amylin (100 nM or 10 µM) and the selective amylin receptor antagonist, AC-187 [13], [62]–[65] (1–100 nM) for 24 hours. Immunocytochemistry revealed a dose-dependent inhibition of amylin monomer uptake and its concomitant accumulation on the PM in RIN-m5F cells at low (100 nM) human amylin concentration (Figure 1A, top panel and graph) and in human islets (Figure S1A, top panel and graph) indicating a receptor-dependent mechanism in both cell types. When a high 10 µM amylin concentration was used, the extent of human amylin monomer internalization was not significantly changed by AC-187 in RIN-m5F cells (Figure 1A, bottom panel and graph) or in human islets (Figure S1A, bottom panel, and graph), suggesting an AM-R-independent uptake mechanism. Thus, our results indicate that the mechanism of amylin monomer internalization is dependent on its concentration.

Bottom Line:
In contrast a majority of the oligomers at both early (1 hour) and late times (24 hours) traffic with a fluid-phase marker, dextran, to the same endocytotic compartments, the uptake of which is blocked by potent macropinocytotic inhibitors.This led to a significant increase in extra-cellular PM accumulation, in turn potentiating amylin toxicity in pancreatic cells.Our studies suggest that macropinocytosis is a major but not the only clearance mechanism for both amylin's molecular forms, thereby serving a cyto-protective role in these cells.

Affiliation:
Department of Biological Sciences, The George Washington University, Washington, District of Columbia, United States of America.

ABSTRACTToxic human amylin oligomers and aggregates are implicated in the pathogenesis of type 2 diabetes mellitus (TTDM). Although recent studies have shown that pancreatic cells can recycle amylin monomers and toxic oligomers, the exact uptake mechanism and trafficking routes of these molecular forms and their significance for amylin toxicity are yet to be determined. Using pancreatic rat insulinoma (RIN-m5F) beta (β)-cells and human islets as model systems we show that monomers and oligomers cross the plasma membrane (PM) through both endocytotic and non-endocytotic (translocation) mechanisms, the predominance of which is dependent on amylin concentrations and incubation times. At low (≤ 100 nM) concentrations, internalization of amylin monomers in pancreatic cells is completely blocked by the selective amylin-receptor (AM-R) antagonist, AC-187, indicating an AM-R dependent mechanism. In contrast at cytotoxic (µM) concentrations monomers initially (1 hour) enter pancreatic cells by two distinct mechanisms: translocation and macropinocytosis. However, during the late stage (24 hours) monomers internalize by a clathrin-dependent but AM-R and macropinocytotic independent pathway. Like monomers a small fraction of the oligomers initially enter cells by a non-endocytotic mechanism. In contrast a majority of the oligomers at both early (1 hour) and late times (24 hours) traffic with a fluid-phase marker, dextran, to the same endocytotic compartments, the uptake of which is blocked by potent macropinocytotic inhibitors. This led to a significant increase in extra-cellular PM accumulation, in turn potentiating amylin toxicity in pancreatic cells. Our studies suggest that macropinocytosis is a major but not the only clearance mechanism for both amylin's molecular forms, thereby serving a cyto-protective role in these cells.